bOur previous study showed that Akt phosphorylates TopBP1 at the Ser-1159 residue and induces its oligomerization. Oligomerization is required for TopBP1 to bind and repress E2F1 activity. However, the mechanism through which phosphorylation of TopBP1 by Akt leads to its oligomerization remains to be determined. Here, we demonstrate that binding between the phosphorylated Ser-1159 (pS1159) residue and the 7th and 8th BRCT domains of TopBP1 mediates TopBP1 oligomerization. Mutations within the 7th and 8th BRCT domains of TopBP1 that block binding to a pS1159-containing peptide block TopBP1 oligomerization and its ability to bind and repress E2F1 activities. The Akt-induced TopBP1 oligomerization is also directly demonstrated in vitro by size exclusion chromatography. Importantly, oligomerization perturbs the checkpoint-activating function of TopBP1 by preventing its recruitment to chromatin and ATR binding upon replicative stress. Hyperactivation of Akt inhibits Chk1 phosphorylation after hydroxyurea treatment, and this effect is dependent on TopBP1 phosphorylation at Ser-1159. Thus, Akt can switch the TopBP1 function from checkpoint activation to transcriptional regulation by regulating its quaternary structure. This pathway of regulation is clinically significant, since treatment of a specific Akt inhibitor in PTEN-mutated cancer cells inhibits TopBP1 oligomerization and causes its function to revert from promoting survival to checkpoint activation.

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: (i) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and (ii) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.ormal cells use various fundamental braking mechanisms to properly respond to environmental cues and coordinate the execution of different phases of cell cycle. Tumor suppressors p53 and pRb are the key regulators for G1 checkpoint, which are frequently lost in many types of cancer (1). Mutations of TP53 are found in half of all human cancers, including nearly all smallcell lung cancers, squamous cell lung cancers, high-grade serous ovarian cancer (2-4), and greater than 80% of glioblastoma and basal-like breast cancer (5, 6). Therefore, understanding the contribution of TP53 mutations in carcinogenesis is very important for the development of new strategies to prevent cancer progression and improve the efficacy of cancer therapy.In addition to the loss of normal p53 function, mutant form of p53 (mutp53) proteins acquire new oncogenic properties (gainof-function, GOF), such as promoting cancer cell proliferation, metastasis, genomic instability, resistance to chemotherapy, etc. (7-9). Among the many mechanisms of mutp53 GOF, the checkpoint activator TopBP1 (topoisomerase IIβ-binding protein) has been identified as a critical mediator for facilitating complex formation between several hotspot mutp53 proteins and either NF-Y or p63/p73 (10). TopBP1 interacts with these mutp53s and NF-Y and promotes mutp53 and p300 recruitment to N...

Background: Posttranslational modifications of E2F1 alter its transcriptional activity. Results: UCH37 binds E2F1 and deubiquitinates its UbK63-specific linkages to enhance the transcriptional activation of E2F1 target genes, particularly upon DNA damage. Conclusion: Ubiquitination and deubiquitination of UbK63-specific linkages provide an additional layer of regulation for E2F1 transcriptional activity. Significance: UCH37 is the first known deubiquitinating enzyme to directly regulate E2F1.

Oxidative stress is a ubiquitous threat to all aerobic organisms and has been implicated in numerous pathological conditions such as cancer. Here we demonstrate a pivotal role for E2F1, a cell cycle regulatory transcription factor, in cell tolerance of oxidative stress. Cells lacking E2F1 are hypersensitive to oxidative stress due to the defects in cell cycle arrest. Oxidative stress inhibits E2F1 transcriptional activity, independent of changes in association with Rb and without decreasing its DNA-binding activity. Upon oxidative insult, SUMO2 is extensively conjugated to E2F1 mainly at lysine 266 residue, which specifically modulates E2F1 transcriptional activity to enhance cell cycle arrest for cell survival. We identify SENP3, a desumoylating enzyme, as an E2F1-interacting partner. Oxidative stress inhibits the interaction between E2F1 and SENP3, which leads to accumulation of sumoylated E2F1. SENP3-deficient cells exhibit hypersumoylation of E2F1 and are resistant to oxidative insult. High levels of SENP3 in breast cancer are associated with elevated levels of E2F targets, high tumor grade, and poor survival. Given the prevalence of elevated levels of SENP3 across numerous cancer types, the SENP3-E2F1 axis may serve as an avenue for therapeutic intervention in cancer.

Cdk2-dependent TopBP1-treslin interaction is critical for DNA replication initiation. However, it remains unclear how this association is terminated after replication initiation is finished. Here, we demonstrate that phosphorylation of TopBP1 by Akt coincides with cyclin A activation during S and G2 phases and switches the TopBP1-interacting partner from treslin to E2F1, which results in the termination of replication initiation. Premature activation of Akt in G1 phase causes an early switch and inhibits DNA replication. TopBP1 is often overexpressed in cancer and can bypass control by Cdk2 to interact with treslin, leading to enhanced DNA replication. Consistent with this notion, reducing the levels of TopBP1 in cancer cells restores sensitivity to a Cdk2 inhibitor. Together, our study links Cdk2 and Akt pathways to the control of DNA replication through the regulation of TopBP1-treslin interaction. These data also suggest an important role for TopBP1 in driving abnormal DNA replication in cancer.

Topoisomerase IIβ-binding protein 1 (TopBP1) is involved in cellular replication among other functions and is known to activate ATR/Chk1 during replicative stress. TopBP1 is also expressed at high levels in many cancers. However, the impact of TopBP1 overexpression on ATR/Chk1 activation and cancer development has not been investigated. Here we demonstrate that the degree of ATR/Chk1 activation is regulated by TopBP1 in a biphasic, concentration-dependent manner in a nontransformed MCF10A cell line and several cancer cell lines, including H1299, MDA-MB468, and U2OS. At low levels, TopBP1 activates ATR/Chk1, but once TopBP1 protein accumulates above an optimal level, it paradoxically leads to lower activation of ATR/Chk1. This is due to the perturbation of ATR–TopBP1 interaction and ATR chromatin loading by excessive TopBP1. Overexpression of TopBP1 thus hinders the ATR/Chk1 checkpoint response, leading to the impairment of genome integrity as demonstrated by the cytokinesis-block micronucleus assay. In contrast, moderate depletion of TopBP1 by shRNA in TopBP1-overexpressing cancer cells enhanced ATR/Chk1 activation and S-phase checkpoint response after replicative stress. The clinical significance of these findings is supported by an association between TopBP1 overexpression and genome instability in many types of human cancer. Taken together, our study illustrates an unexpected relationship between the levels of TopBP1 and the final functional outcome and suggests TopBP1 overexpression as a new mechanism directly contributing to genomic instability during tumorigenesis.

BackgroundCGRRF1 is a growth suppressor and consists of a transmembrane domain and a RING-finger domain. It functions as a RING domain E3 ubiquitin ligase involved in endoplasmic reticulum-associated degradation. The expression of CGRRF1 is decreased in cancer tissues; however, the role of CGRRF1 in breast cancer and the mechanism(s) of its growth suppressor function remain to be elucidated.MethodsTo investigate whether CGRRF1 inhibits the growth of breast cancer, we performed MTT assays and a xenograft experiment. Tumors harvested from mice were further analyzed by reverse phase protein array (RPPA) analysis to identify potential substrate(s) of CGRRF1. Co-immunoprecipitation assay was used to verify the interaction between CGRRF1 and its substrate, followed by in vivo ubiquitination assays. Western blot, subcellular fractionation, and reverse transcription quantitative polymerase chain reaction (qRT-PCR) were performed to understand the mechanism of CGRRF1 action in breast cancer. Publicly available breast cancer datasets were analyzed to examine the association between CGRRF1 and breast cancer.ResultsWe show that CGRRF1 inhibits the growth of breast cancer in vitro and in vivo, and the RING-finger domain is important for its growth-inhibitory activity. To elucidate the mechanism of CGRRF1, we identified EGFR as a new substrate of CGRRF1. CGRRF1 ubiquitinates EGFR through K48-linked ubiquitination, which leads to proteasome degradation. In addition to regulating the stability of EGFR, knockout of CGRRF1 enhances AKT phosphorylation after EGF stimulation. By analyzing the breast cancer database, we found that patients with low CGRRF1 expression have shorter survival. As compared to normal breast tissues, the mRNA levels of CGRRF1 are lower in breast carcinomas, especially in HER2-positive and basal-like breast cancers. We further noticed that CGRRF1 promoter methylation is increased in breast cancer as compared to that in normal breast tissue, suggesting that CGRRF1 is epigenetically modified in breast cancer. Treatment of 5-azactidine and panobinostat restored CGRRF1 expression, supporting that the promoter of CGRRF1 is epigenetically modified in breast cancer. Since 5-azactidine and panobinostat can increase CGRRF1 expression, they might be potential therapies for breast cancer treatment.ConclusionWe demonstrated a tumor-suppressive function of CGRRF1 in breast cancer and identified EGFR as its target.

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